Our long-term goal is to characterize tumor antigen-specific T-cell responses in melanoma patients and identify the T-cell properties that correlate best with clinical efficacy. We have employed a model of gp100209-2M vaccination in the adjuvant setting to evaluate various immune monitoring strategies. We have shown that 33 of 35 stage I-III melanoma patients demonstrated significant increases in the percentage of tetramer+ CD8+ T cells (mean 0.36%; range 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry (CFC-?FN?) analysis after brief peptide stimulation in vitro demonstrated that most, but not all tetramer+ T cells produced IFN-gamma. A substantial fraction of tetramer+ CD8+ T cells (up to 50% in some patients) failed to produce IFNgamma- suggesting functional anergy. Vaccine-induced, peptide-specific T cells recognized the native gp100209-2M peptide and were able to kill A2+ gp100+ melanoma cell lines. The gp100-specific response in vaccinated patients waned over time, but low numbers of highly cognate-antigen responsive tetramer+ memory CD8+ T cells persisted 12-26 months following their last vaccination. The objective of this application is to characterize the gp100 peptide T-cell memory response in patients free of disease > 12 months after their last gp100(209-2M) peptide vaccination. Our central hypothesis is that previously vaccinated patients will have CD8+ T cells with a heightened functional and proliferative potential and that revaccination will restore (or even increase) the frequency of gp100 peptide-specific T cells to frequencies present at the completion of the original vaccination regimen. The specific aims of the study are to 1) determine the kinetics and duration of the gp100209-2M-specific CD8+ T-cell memory response after booster vaccination; 2) determine the potency of the gp100(209-2M)-specific T-cell memory response after booster vaccination and 3) characterize the cell surface phenotype and function of memory/effector CD8+ T cells after booster immunization. The results of this study may provide new information relevant to the improvement of therapeutic melanoma vaccines.